The specific objective of this project is on the cell and molecular biological mechanisms regulating skeletal development and mesenchymal cell differentiation, specifically the action of growth factors. A summary of our research progress s presented below: 1. Spatiotemporal protein distribution of TGF-betas, their receptors, and extracellular matrix molecules during embryonic tendon development Tendon is one of the least understood tissues of the musculoskeletal system in terms of development and morphogenesis. Collagen fibrillogenesis has been the most studied aspect of tendon development, focusing largely on the role of matrix molecules such as collagen type III and decorin. While involvement of matrix molecules in collagen fibrillogenesis during chick tendon development is well understood, the role of growth factors has yet to be elucidated. This work examines the expression patterns of TGF-beta1, -2, and -3, and their receptors with respect to expression patterns of collagen type III, decorin, and fibronectin. We focus on the intermediate stages of tendon development in the chick embryo, a period during which the tendon micro- and macro-architecture are being established. Our findings demonstrate for the first time that TGF-beta1, -2, and -3 have distinct spatiotemporal developmental protein localization patterns in the developing tendon and strongly suggest that these isoforms have independent roles in tendon development. 2. Interaction of cartilage oligomeric matrix protein/thrombospondin 5 with aggrecan Cartilage oligomeric matrix protein (COMP), also known as thrombospondin 5 (TSP5), is a pentameric extracellular matrix protein. Based on its sequence COMP/TSP5 belongs to the TSP family (1,2). It is a major component of the extracellular matrix (ECM) of the musculoskeletal system including cartilage, tendon, and ligament. The physiological function of COMP/TSP5 remains largely unclear, but its importance is underscored by its association with several joint diseases. To study the functions of COMP/TSP5 in cartilage ECM, we have examined its interaction with glycosaminoglycans (GAGs) and aggrecan, a major and critical components of cartilage extracellular matrix. We also tested MUT3, a COMP/TSP5 mutant found to account for 30% of human pseudoachondroplasia cases, to determine if the mutation affects the function of the protein. Using affinity co-electrophoresis (ACE), we show that COMP/TSP5 bound to heparin, chondroitin sulfates, and heparan sulfate in its native calcium-replete conformation. EDTA treatment of COMP/TSP5 reduces heparin binding and inhibits chondroitin sulfate binding, implying a conformation-dependent GAGbinding site in the molecule. MUT3 showed weaker binding to the GAGs than COMP/TSP5 in the presence of Ca, and EDTA treatment of MUT3 further decreased binding. Using a solid binding assay, we showed that COMP/TSP5 can bind aggrecan, which is decreased upon EDTA chelation, in agreement with the ACE data. MUT3 showed weak interaction with aggrecan. Also, soluble GAGs inhibited aggrecan binding to COMP/TSP5, suggesting that the interaction of aggrecan with COMP/TSP5 was through binding to its GAG side chains. Using recombinant COMP/TSP5 fragments covering various COMP/TSP5 domains were tested, the C-globe region alone was found to bind to aggrecan in solution, suggesting that this domain can mediate the interaction of COMP/TSP5 and aggrecan. These data show that COMP/TSP5 is a proteoglycan-binding protein, and it binds aggrecan, possibly through the GAg side chains on aggrecan and the C-globe regain of COMP/TSP5. This interaction is regulated by calcium-binding sensitiv conformation of the molecule. Our results suggest that COMP/TSP5 may function to support matrix interaction in the extracellular matrix of cartilage.